Cell proliferation reagent wst 1tm

Two mammalian cell lines, including human embryonic kidney cells (HEK293T) and human breast cancer cells (MCF7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone^TM, Logan, UT, United States) supplemented with 10% FBS (Hyclone^TM) and 1% penicillin/streptomycin (Hyclone^TM) and incubated at 37°C and 5% CO₂ up to 80% confluence. Cellular adherence to the substratum was disrupted using Accutase (Innovative Cell Technologies, San Diego, CA, United States). In total, 1 × 10⁴ to 4 × 10⁴ cells in each well of a 96-well plate containing 8, 16, 32, and 64 μg/mL of ModoCath peptides were incubated for 24 h at 37°C and 5% CO₂. Additionally, HEK293T cells were incubated in the FBS-free medium. Triton X 100 (Sigma Aldrich) was used as a positive control for complete cell lysis, and untreated cells were used as the negative control. After incubation, the medium was removed from the wells, and 10 μL of coloring solution (Cell Proliferation Reagent WST-1^TM; Sigma Aldrich) and 100 μL of DMEM (Hyclone^TM) were added to the wells in accordance with the manufacturer’s protocol. Absorbance was measured for each well at 440 nm (peptide-treated and control) and 650 nm (background) and recorded as the OD, using a microplate reader (xMark^TM spectrophotometer; Bio-Rad). Cell viability was calculated using the following equation:
All experiments were performed in triplicate.

HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone^TM, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone^TM) and 1% penicillin/streptomycin (Pen-Strep; Hyclone^TM) up to 80% confluence at 37 ℃ and 5% CO₂. Cells were detached from the plate by adding 0.25% trypsin-EDTA solution (Gibco^TM; Carlsbad, CA, USA). In a 96-well plate, 2 × 10⁴ cells were seeded per well and cultured for 24 h. Subsequently, the medium was replaced with 100 μL DMEM (Hyclone^TM) containing 10% heat-inactivated FBS. Peptides were added to each well at concentrations of 64 μg/mL and 160 μg/mL. As references for complete cell lysis, 64 μg/mL melittin (Sigma-Aldrich) and Triton X-100 (Sigma-Aldrich) were used. Untreated cells were used as negative controls. After 24 h incubation at 37 ℃ and 5 % CO₂, 10 μL of coloring solution (Cell Proliferation Reagent WST-1^TM; Sigma Aldrich) was added to each well according to the manufacturer’s instructions. The absorbance at 450 nm (treated group and control) and 650 nm (background) was measured in each well using a microplate reader (xMarkTM spectrophotometer; Bio-Rad). The cell viability was calculated using the following equation. All experiments were performed in triplicate.